Protein-capture reagents are indispensable for delineating the molecular mechanisms of diseases, to detect and characterize cellular abnormalities, and to characterize biological effects of drugs. However, the current paucity of high-quality protein-capture reagents presents a major bottleneck in virtually all areas of biomedical sciences. The overarching goal of this project is to develop an innovative and powerful protein-capture technology with high levels of fidelity and predictability. We aim to overcome a major limitation of currently available technologies where specificity and epitopes must be individually tested by laborious methods after generating protein-capture reagents. We introduce a new concept, "C-clamping", that enables to direct capture reagents ("C-clamps") exclusively to the C-terminal 6-8 residues of proteins with high fidelity and high affinity. C-clamps are in the form of robust recombinant binding proteins generated using state- of-the-art phage-display technologies. Virtually every protein has a unique C-terminal signature that can be recognized with high efficiency by C-clamps. The a priori knowledge of epitope location allows one to accurately predict the level of specificity by identifying potential cross- reactivity through database search and to implement strategies to eliminate off-targets. These attributes make C-clamps particularly suited as the core technology for generating a comprehensive set of protein capture reagents. Furthermore, C-clamping is ideally suited to detect proteolytic "neo-epitopes" generated by proteolysis, markers of biomedically important processes (e.g. apoptosis). Our proof-of-concept experiments have successfully demonstrated the feasibility and enormous potential of C-clamping. Proposed studies aim to establish C-clamping as a general technology by producing high- performance capture reagents for high-value targets including integral membrane proteins, splice variants, viral proteins and caspase neo-epitopes. C-clamping represents a paradigm shift in capture-reagent generation, and the establishment of C-clamping will make large contributions to the entire molecular biomedical sciences. PUBLIC HEALTH RELEVANCE: Detecting and measuring the amounts of proteins are critical for understanding differences between normal and diseased states of cells and tissues. This project will establish a totally new approach to facile generation of detection reagents that are high performance, easy to produce and easy to made available to the research community. This innovative and powerful technology will fill a major void in the currently available molecular tools and will have a major impact on virtually all areas of molecular biomedical sciences, diagnosis and drug development.